CN101659707B - Method for extracting and separating D-fraction polysaccharide from fruit bodies or mycelia of polyporus frondosus - Google Patents
Method for extracting and separating D-fraction polysaccharide from fruit bodies or mycelia of polyporus frondosus Download PDFInfo
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Abstract
The invention relates to a method for extracting and separating D-fraction polysaccharide from fruit bodies or mycelia of polyporus frondosus, which is characterized in that: the fruit bodies or mycelia of the polyporus frondosus is crushed, extracted by hot water, precipitated by ethanol to prepare crude polysaccharide; and the crude polysaccharide is subjected to complex reaction, acid pickling, and deproteinization by a sevag method, and then is purified step by step through graded ethanol precipitation to prepare the target product, namely the D-fraction polysaccharide of the polyporus frondosus. The crude polysaccharide is obtained by a water extraction and ethanol precipitation method, and then is separated and purified according to different natures of a complex compound and the D-fraction polysaccharide to the PH condition. The method has convenient operation, obtains the target product with high purity, and provides a feasible way for producing the D-fraction polysaccharide of the polyporus frondosus on a large scale, and preparing various medicaments and health-care foods.
Description
Technical field
The present invention relates to the extraction and separation method of edible mushrooms effective constituent, be specifically related to the extraction and separation method of a kind of Grifola Frondosa sporophore or mycelium polysaccharides component.
Background technology
Grifola frondosa (Polyporus frondosus) is under the jurisdiction of Mycophyta, Basidiomycotina, Aphyllophorales, polyporaceae, Polyporus.Grifola frondosa is famous edible, medicinal fungi, and is nutritious, tasty, is the macro fungi of " but edible, can mend medicine, the whole body is precious ".Grifola frondosa property is flat, flavor is sweet, can be used for treating diseases such as dysuria, oedema, beriberi, hepatic ascites, mellitus, hypertension and obesity.
Known mycelium or sporophore from the Grifola frondosa Pseudomonas extract by having β-1; The 3-key connects the β-1 of branched glucose; The 6-key connects that the glucose main chain constitutes or by having β-1, the 6-key connects the β-1 of branched glucose, and the polysaccharide that the 3-key connects glucose main chain formation has antitumour activity.
It also is known comprising the following steps to prepare cancer-resisting substance, and said step is: with hot water to Grifola frondosa, tree flower bacterium department resemble ear orchid (Grifola gigantea) (Tonbimai) or Laetiporus sulphureus (Masutake) extract; The concentrating under reduced pressure extracting solution precipitates liquid concentrator with organic solvent, throw out is dialysed removing low-molecular-weight material, and with lipophilic organic solvent extraction impurity from dialyzate, to remove them.
Yet; Those methods of more than describing are for a large amount of medicament of preparation and to come to prepare effectively for the heath food from limited resources be to be not suitable for; Because the purification step of these methods is quite complicated, and contains the material that suppresses immune-enhancing activity in the product of gained.
Summary of the invention
Technical problem to be solved by this invention is to utilize the different properties of Grifola frondosa D fraction polysaccharide in different ethanol concentration, makes Grifola Frondosa sporophore or mycelium D component obtain the fractional separation purifying, and a kind of method of effective classification purification D fraction polysaccharide is provided.
The present invention realizes that the technical scheme of Grifola Frondosa sporophore or mycelium D fraction polysaccharide fractional separation purifying is:
With raw material pulverizing, hot water extraction, ethanol sedimentation adds the complex compound complexing, pickling, alkali redissolution sevag method deproteinated, ethanol sedimentation, alkali redissolves, and ethanol precipitates once more, and alcohol is washed, lyophilize.Remember grifolan.
Separating and purifying technology of the present invention is achieved through following steps:
A. the extraction of Crude polysaccharides
With Grifola Frondosa sporophore or mycelium is raw material, is ground into fine powder, adds 10～40 times water; Hot water extraction 1-6 hour, centrifugal, being evaporated to the liquid concentrator specific density was 1.02～1.15; Slowly ethanol to the final concentration of adding 95% is 30%～70%, stirs 4 ℃ of hold over night; Filter, precipitate very Grifola frondosa Crude polysaccharides.
The grading purification of B.D fraction polysaccharide
(1) the gained Crude polysaccharides is extracted ethanol among the A, redissolves with zero(ppm) water;
(2) CTAOH of adding 70% regulates PH >=10,4 ℃ maintenance 24-72h to no longer producing deposition, spinning, and abandoning supernatant must precipitate;
(3) get above-mentioned (2) gained deposition and add acetum washing 3 times, centrifugal, 80% washing with alcohol 3 times, centrifugal, the gained deposition is redissolved with the NaOH solution of 0.1～1M, and centrifugal, supernatant is with sevag method deproteinated;
(4) solution behind (3) deproteinated being added ethanol to alcohol concn is 40%～60%, leaves standstill, centrifugal, and deposition is redissolved with NaOH solution; Adding ethanol to final concentration is 30%～40%, leaves standstill, centrifugal, gets deposition and redissolves the back and add ethanol to make alcohol concn be 20%～30%; Leave standstill, centrifugal, the collecting precipitation thing; Ethanol with 95% and absolute ethanol washing, lyophilize promptly gets Grifola frondosa D fraction polysaccharide.
Above-mentioned cryodesiccated program is :-47 ℃ of 2h, and-30 ℃ of 4h ,-5 ℃ of 24h, 27 ℃ of 4h, vacuum tightness remains on 0.08MPa～0.09Mpa.
The polysaccharide of D component be can with the protein-polysaccharide of CTA-group set, aggregation is water insoluble, wherein sugared content is about 90%, protein contnt 6%--10%, molecular weight are 80～1,200,000 dalton and have good antineoplastic activity.
The present invention utilizes water to carry ethanol precipitation and obtains Crude polysaccharides, utilizes complex compound and D fraction polysaccharide to make it obtain separation and purification to ethanol alcohol precipitation concentration different properties again.The present invention is easy to operate, and gained title product purity is high, is the scale operation of performance Grifola frondosa D fraction polysaccharide, and preparing various medicines and protective foods provides feasible approach.
Embodiment
The extraction of embodiment 1 Grifola frondosa Crude polysaccharides
Take by weighing Grifola Frondosa sporophore or mycelium fine powder 15Kg, add 500L water, 100 ℃ are extracted 1h; The centrifugal 20min of 8000rpm, supernatant concentration to density is 1.04, adds 95% ethanol of 3 times of amounts; 4 ℃ of hold over night are filtered, and gained is deposited in to preserve in the small amount of ethanol and has.
The separation and purification of the grifolan D component of embodiment 2 90 ten thousand Dalton molecular weights
Grifolan medicinal extract adds 30 times hot water redissolution, and is centrifugal, and the PTOH of adding 30% extremely no longer produces deposition, adjusting PH=11, and 4 ℃ of maintenance 48h, spinning, abandoning supernatant must precipitate; Deposition is with 20% the acetic acid washing of 2 times of volumes 2 times, and 90% washing with alcohol 2 times is with 0.5 NaOH solution redissolution, centrifugal again, and supernatant is with sevag method deproteinated 3 times; It is 55% to make deposition that supernatant adds ethanol to alcohol concn, and deposition is redissolved with the NaOH solution of 0.2M, and adding ethanol, to make alcohol concn be 35%, precipitates; Centrifugal, get NaOH solution that deposition uses 0.2M and redissolve the back and add ethanol to make alcohol concn be 30%, deposition, centrifugal; Get deposition and wash respectively with absolute ethyl alcohol with 90%, lyophilize obtains 4.5g Grifola frondosa D fraction polysaccharide; Polysaccharide content is 91%, and protein content is 6.67%, and molecular weight is 90W dalton.
The separation and purification of the grifolan D component of embodiment 3 115 ten thousand Dalton molecular weights
Grifolan medicinal extract adds 30 times hot water redissolution, and is centrifugal, and the CTAOH of adding 30% extremely no longer produces deposition, adjusting PH=12, and 4 ℃ of maintenance 48h, spinning, abandoning supernatant must precipitate; Deposition is with 20% the acetic acid washing of 2 times of volumes 2 times, and 90% washing with alcohol 2 times is with 0.5 NaOH solution redissolution, centrifugal again, and supernatant is with sevag method deproteinated 3 times; It is 40% to make deposition that supernatant adds ethanol to alcohol concn, and deposition is redissolved with the NaOH solution of 0.2M, and adding ethanol, to make alcohol concn be 30%, precipitates; Centrifugal, get NaOH solution that deposition uses 0.2M and redissolve the back and add ethanol to make alcohol concn be 25%, deposition, centrifugal; Get deposition and wash respectively with absolute ethyl alcohol with 90%, lyophilize obtains 3.2g Grifola frondosa D fraction polysaccharide; Polysaccharide content is 93%, and protein content is 5.61%, and molecular weight is 115W dalton.
The separation and purification of the grifolan D component of embodiment 4 100 ten thousand Dalton molecular weights
Grifolan medicinal extract adds 30 times hot water redissolution, and is centrifugal, and the CTAOH of adding 40% extremely no longer produces deposition, adjusting PH=11, and 4 ℃ of maintenance 48h, spinning, abandoning supernatant must precipitate; Deposition is with 20% the acetic acid washing of 2 times of volumes 2 times, and 90% washing with alcohol 2 times is with 0.5 NaOH solution redissolution, centrifugal again, and supernatant is with sevag method deproteinated 3 times; It is 50% to make deposition that supernatant adds ethanol to alcohol concn, and deposition is redissolved with the NaOH solution of 0.2M, and adding ethanol, to make alcohol concn be 25%, precipitates; Centrifugal, get NaOH solution that deposition uses 0.2M and redissolve the back and add ethanol to make alcohol concn be 30%, deposition, centrifugal; Get deposition and wash respectively with absolute ethyl alcohol with 90%, lyophilize obtains 3.9g Grifola frondosa D fraction polysaccharide; Polysaccharide content is 91.5%, and protein content is 6.02%, and molecular weight is 100W dalton.
Claims (3)
1. the extraction and separation method of Grifola Frondosa sporophore or mycelium D fraction polysaccharide is characterized in that this method is that Grifola Frondosa sporophore or mycelium are pulverized hot water extraction; Ethanol sedimentation; Obtain Crude polysaccharides, carry out complex reaction, pickling then; With classification alcohol precipitation purifying progressively, obtain title product grifolan D component behind the sevag method deproteinated;
The separation of wherein said protein-polysaccharide comprises the following steps:
(1) be raw material with Grifola Frondosa sporophore or mycelium, add water extraction after being ground into fine powder, centrifugal, concentrating under reduced pressure, the liquid concentrator alcohol precipitation leaves standstill, and filters, and deposition is the Grifola frondosa Crude polysaccharides;
(2) CTAOH with gained Grifola frondosa Crude polysaccharides adding 70% in (1) precipitates to no longer producing, and regulates pH >=10, leaves standstill, and is centrifugal, gets deposition;
(3) get above-mentioned (2) gained deposition with acetum washing 3 times, centrifugal, deposition is with 80% washing with alcohol 3 times, and centrifugal, the gained deposition is with the redissolution of NaOH solution, and centrifugal, supernatant is with sevag method deproteinated;
(4) solution behind the deproteinated in (3) being added ethanol to alcohol concn is 40%～60%, leaves standstill, centrifugal, and deposition is redissolved with NaOH solution; Adding ethanol to final concentration is 30%～40%, leaves standstill, centrifugal, gets deposition and redissolves the back and add ethanol to make alcohol concn be 20%～30%; Leave standstill, centrifugal, the collecting precipitation thing; Ethanol with 95% and absolute ethanol washing, lyophilize promptly gets Grifola frondosa D fraction polysaccharide.
2. the extraction and separation method of Grifola Frondosa sporophore as claimed in claim 1 or mycelium D fraction polysaccharide is characterized in that wherein said:
Said cryodesiccated program is :-47 ℃ of 2h, and-30 ℃ of 4h ,-5 ℃ of 24h, 27 ℃ of 4h, vacuum tightness remains on 0.08MPa～0.09Mpa.
3. the extraction and separation method of Grifola Frondosa sporophore as claimed in claim 1 or mycelium D fraction polysaccharide is characterized in that wherein said:
The D fraction polysaccharide be can with the protein-polysaccharide of CTA-group set, water insoluble, wherein polysaccharide content is 90%, molecular weight is 80～1,200,000 dalton.
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| CN102080113B (en) * | 2010-12-09 | 2013-04-17 | 江苏大学 | Method for producing polysaccharide by rice husk bran composite raw material and grifola frondosa mutant strain |
| CN104277129A (en) * | 2013-07-01 | 2015-01-14 | 周伯扬 | Method for extracting grifola frondosa polysaccharides |
| CN103833866B (en) * | 2013-12-23 | 2015-10-28 | 浙江老艺人生物科技有限公司 | A kind of method extracting grifolan based on ultrasonic wave-vacuum impregnation assist efficient |
| US20170312324A1 (en) * | 2014-10-28 | 2017-11-02 | Yunnan University | Use of maitake and maitake grifolan d component in preparation of antidepressant drug |
| CN105285284A (en) * | 2015-11-25 | 2016-02-03 | 迁西县尚菌堂生物科技有限公司 | Grifola frondosa domestic fungus pressed candy and making method thereof |
| CN105434344B (en) * | 2016-01-08 | 2019-04-05 | 福建农林大学 | A kind of anti-enterovirns type 71 oral solution of grifolan and preparation method thereof |
| CN105777931A (en) * | 2016-03-02 | 2016-07-20 | 漳州片仔癀药业股份有限公司 | Grifolan extracting method and pharmaceutical application thereof |
| CN105732836B (en) * | 2016-03-02 | 2018-11-13 | 漳州片仔癀药业股份有限公司 | The extraction process of grifolan and its purposes on preparing the drug for repairing gastric mucosa wound |
| CN105669876A (en) * | 2016-03-02 | 2016-06-15 | 漳州片仔癀药业股份有限公司 | Extraction process and application of grifolan |
| CN114316080B (en) * | 2021-11-17 | 2023-04-07 | 浙江工商大学 | Method for improving extraction rate and bioactivity of grifola frondosa crude polysaccharide |
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Owner name: ZHEJIANG FANGGE PHARMACEUTICAL CO., LTD. Free format text: FORMER OWNER: XU CAIQUAN Effective date: 20121022 |
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Effective date of registration: 20121022 Address after: 323800 Zhejiang city of Lishui province Qingyuan County Star Road Garden Bridge Patentee after: Zhejiang Fangge Pharmaceutical Co., Ltd. Address before: 323800 Zhejiang province Qingyuan County Star Road Garden Bridge No. 98 Patentee before: Xu Caiquan |